Mar 23, 2018 - ficity protein 1 (SP1), ETS proteins such as ETV2, FLI1, ERG, and ETS11,2, ... Robo4 is a transmembrane protein that stabilizes vasculature in ...
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Received: 18 October 2017 Accepted: 23 March 2018 Published: xx xx xxxx
ETV2-TET1/TET2 Complexes Induce Endothelial Cell-Specific Robo4 Expression via Promoter Demethylation Toru Tanaka 1, Kohei Izawa1, Yusuke Maniwa1, Maki Okamura1, Atsumasa Okada2, Tomoko Yamaguchi2, Keisuke Shirakura1, Naoki Maekawa1, Hayato Matsui1, Kenji Ishimoto1, Nobumasa Hino1, Osamu Nakagawa3, William C. Aird4, Hiroyuki Mizuguchi1, Kenji Kawabata2, Takefumi Doi1 & Yoshiaki Okada1 Although transcription factors regulating endothelial cell (EC)-specific gene expression have been identified, it is not known how those factors induce EC-specificity. We previously reported that DNA hypomethylation of the proximal promoter elicits EC-specific expression of Roundabout4 (Robo4). However, the mechanisms establishing EC-specific hypomethylation of the Robo4 promoter remain unknown. In this study, we demonstrated that the hypermethylated Robo4 proximal promoter is demethylated as human iPS cells differentiate into endothelial cells. Reporter assays demonstrated that ETV2, an ETS family transcription factor, bound to ETS motifs in the proximal promoter and activated Robo4 expression. Immunoprecipitation demonstrated direct interaction between ETV2 and methylcytosine-converting enzymes TET1 and TET2. Adenoviral expression of ETV2-TET1/TET2 complexes demethylated the Robo4 promoter and induced Robo4 expression in non-ECs. In summary, we propose a novel regulatory model of EC-specific gene expression via promoter demethylation induced by ETV2-TET1/TET2 complexes during endothelial differentiation. To determine the underling mechanisms of tissue-specific gene expression, various genes have been studied. Endothelial cell (EC)-specific genes have been shown to be regulated by transcription factors, including specificity protein 1 (SP1), ETS proteins such as ETV2, FLI1, ERG, and ETS11,2, Group F Sry-related high-mobility box factors (SOX7, −17, and −18), and vascular endothelial zinc finger 13. Among these, ETV2 is essential for development of EC and hematopoietic cells4,5 and directly reprograms fibroblasts into ECs6,7. SOX F and VEZF1 in progenitor cells regulate EC function during embryogenesis8,9. Although these factors have been shown to play essential roles during EC differentiation, it remains unclear whether these regulate EC-specific gene expression. Recent reports demonstrate that tissue-specific gene expression is regulated via epigenetic mechanisms, including DNA methylation10. In vertebrates, methylation is catalyzed by DNA methyltransferase, which transfers a methyl group to the C-5 atom of cytosine in a CpG dinucleotide to facilitate gene suppression in cellular processes such as X chromosome inactivation 11. Conversely, DNA demethylation induces transcription12,13, and is regulated by ten-eleven translocation 1–3 (TET1-3), which oxidizes 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. These intermediates are then converted to unmodified cytosine by active or passive demethylation mechanisms14,15. Additionally, transcription factors such as PPARγ, NANOG, PRDM14, and PU.1 were recently demonstrated to directly or indirectly interact with TET1 and/or TET2 to elicit demethylation16–19. In line with this model, the proximal promoters of several EC-specific
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Graduate School of Pharmaceutical Sciences, Osaka University, Suita City, Osaka, 565-0871, Japan. 2Laboratory of Stem Cell Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki City, Osaka, 5670085, Japan. 3Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita City, Osaka, 565-8565, Japan. 4Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA, 02215, USA. Toru Tanaka and Kohei Izawa contributed equally to this work. Correspondence and requests for materials should be addressed to Y.O. (email: okadabos@phs. osaka-u.ac.jp) Scientific RepOrts | (2018) 8:5653 | DOI:10.1038/s41598-018-23937-8
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Figure 1. Demethylation of the Robo4 promoter during differentiation of iPS cells into ECs. (A) Differentiation of human iPS cells into ECs. iPS cells were differentiated into pre-mature (pre-iECs) and mature ECs (iECs). (B) Expression of EC-specific genes in iPS-derived cells. Expression of CD31, VE-cadherin and Robo4 mRNA in iPS cells, pre-iECs, and iECs were measured by real-time RT-PCR. Data are means ± S.D. (n = 3). (C) Methylation patterns of the Robo4 promoter in iPS-derived cells. CpG methylation of the Robo4 promoter in iPS cells, pre-iECs, and iECs was analyzed by bisulfite sequencing. Each graph indicates the CpG position in the promoter and the percentage of methylated CpG.
genes are hypomethylated in ECs, but hypermethylated in non-ECs20. However, the mechanisms by which these promoters are specifically hypomethylated in ECs has not been established. To investigate the mechanisms of EC-specific gene expression, we have been studying an EC-specific gene, Roundabout4 (Robo4)21. Robo4 is a transmembrane protein that stabilizes vasculature in pathological angiogenesis by suppressing EC migration, proliferation, and hyperpermeability induced by vascular endothelial growth factor (VEGF)22–24. Recently, Robo4 has been shown to regulate cytokine production in inflammation25. Robo4 expression is driven by a 3 kb promoter activated by transcription factors such as GA-binding protein (GABP), SP1, AP-1, NF-κB, SOX7, and SOX1826–30. The Robo4 proximal promoter is hypomethylated in ECs and hypermethylated in non-ECs31. This hypermethylation suppresses Robo4 expression by inhibiting SP1 binding to the proximal promoter, and thereby helps restrict expression to ECs, indicating that EC-specific Robo4 expression is regulated by DNA methylation. However, it remains unclear how the Robo4 proximal promoter is specifically demethylated in ECs. In this study, we investigated how methylation of the endogenous Robo4 promoter in human induced pluripotent stem (iPS) cells is altered during differentiation into ECs. We demonstrate that the highly methylated Robo4 promoter is demethylated during cell differentiation and that this demethylation is regulated by ETV2-TET1/ TET2 complexes. Based on these data, we propose a novel regulatory mechanism of EC-specific gene expression.
Results
Robo4 Promoter Is Demethylated During Differentiation of iPS Cells into ECs. To investigate methylation of the human Robo4 promoter, human iPS cells were differentiated into pre-mature (pre-iECs) and mature ECs (iEC) (Fig. 1A). Real-time RT-PCR of transcripts from these cells showed a gradual increase of EC markers, including CD31, VE-cadherin and Robo4, as iPS cells differentiated into ECs (Fig. 1B). We then isolated genomic DNA from these cells, and analyzed methylation of the Robo4 promoter by bisulfite sequencing (Fig. 1C, Supplementary Fig. S1). In iPS cells, the promoter was highly methylated throughout. However, regions within −1.5 kb of the transcription start site were almost completely demethylated in pre-iECs, with the exception of sites at −826 and −756. Further demethylation of sequences between −2906 and −2735 was observed in iECs. Collectively, these data demonstrated that the Robo4 promoter is demethylated at specific positions during differentiation. ETV2 Potentially Demethylates the Robo4 Proximal Promoter. Since the Robo4 promoter is
demethylated as iPS cells differentiate into pre-iECs, we investigated by real-time RT-PCR the expression of transcription factors during differentiation, including SOX7, SOX17, SOX18, VEZF1, and ETV2 (Fig. 2A). Expression
Scientific RepOrts | (2018) 8:5653 | DOI:10.1038/s41598-018-23937-8
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Figure 2. Effect of transcription factors on Robo4 promoter activity in iPS-derived cells. (A) Expression of transcription factors in iPS-derived cells. Expression of SOX factors, VEZF1, and ETV2 in iPS-derived cells was measured by real-time RT-PCR. Data are means ± S.D. (n = 3). (B) Effects of transcription factors on Robo4 promoter activity. HEK293 cells were co-transfected with a Robo4 promoter-luciferase reporter construct and expression vectors for transcription factors or GFP, and luciferase activity was measured 24 h after transfection. Data are means ± S.D. (n = 3). *p
